[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-FEB-16 to 2022-APR-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

Official Journal of the European Union No. L142, May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guideline OPPTS 870.3050: Repeated dose 28-day oral toxicity study in rodents,

Version / remarks:

July 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guideline OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test,

Version / remarks:

July 2000

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guideline OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Petroleum diesel/gas oil fraction, co-processed with renewable hydrocarbons derived from thermally cracked plastics

IUPAC Name:

Petroleum diesel/gas oil fraction, co-processed with renewable hydrocarbons derived from thermally cracked plastics

Test material form:

liquid

Remarks:

Light Yellow

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The CRO has general and reproduction/ developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males: 10-11 weeks; Females: 13-14 weeks
- Weight at study initiation: Males: 291- 340 g; Females: 206 - 261 g
- Housing: On arrival and during the pre-test (females only) and pre-mating period animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).

During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).

During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).

During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.

During locomotor activity monitoring, F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Cages contained sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Sohne GmbH + CO. KG, Rosenberg Germany) equipped with water bottles.

- Diet: pellets (SM R/M-Z from SSNIFF® Spezialdiäten GmbH (Soest, Germany) ad libitum except during designated procedures. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Municipal tap water via water bottles ad libitum. During motor activity measurements, animals did not have access to fresh water for a maximum of 2 hours.
- Acclimation period: 7 days prior to start of the pretest period (females) or 7 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants were provided by the supplier. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water was performed and it was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Targeted: 18 to 24°C; Actual mean: 20 to 22°C
- Humidity (%): Targeted: 40 to 70%; Actual: 23 to 55%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: 2022-JAN-26 (Females); 2022-FEB-09 (Males) TO 2022-APRIL-22

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.

Vehicle:

corn oil

Remarks:

Sigma-Aldrich (Steinheim, Germany). Specific Gravity: 0.92

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Test material dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test material. No correction was made for the purity/composition of the test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil; Trial preparations (not part of thi study) were performed to select the suitable vehicle and to establish a suitable formulation procedure.

- Concentration in vehicle: 0, 25, 75, 250 mg/mL for the control, 100, 300, and 1000 mg/kg/day dose groups, respectively
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration and Homogeneity Analysis:

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20296912).

Concentration analysis:

Dose formulation samples (2 x 500 mg) were collected (from approximately Middle of the dosing container) for analysis from all dose groups during Week 1 of treatment. Mean sample concentration results within or equal to ± 10% of theoretical concentration were considered acceptable.


Homogeneity analysis:
Dose formulation samples (2 x 500 mg) were collected (from approximately Top, Middle, and Bottom of dosing container) for analysis from dose groups 2 and 4 during Week 1 of treatment.


Relative standard deviation (RSD) of concentrations of ≤ 10% for each group were considered acceptable.

Duration of treatment / exposure:

Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy.

Females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 - Control (Corn oil)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 - Low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3 - Mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4 - High dose

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10 Day Dose Range Finder with oral gavage administration of Petroleum diesel/gas oil fraction, co-processed with renewable hydrocarbons derived from thermally cracked plastics (EC 941-803-4) in rats (Test Facility Reference No. 20296913), and in an attempt to produce graded responses to the test material.

- Rationale for animal assignment (if not random): Animals were randomly assigned to groups at arrival. Males and females were randomized separately.

- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours. F0-females were not fasted

- Dose range finding studies: In the 10-day dose range finder (Test Facility Reference No. 20296913), performed with dose levels of 300 and 1000 mg/kg/day, no clinical signs were noted. No relevant changes were noted in body weights and food consumption. No macroscopic findings were noted. Increased liver weights were noted in 1/3 animals at 300 mg/kg/day and in 3/3 animals at 1000 mg/kg/day. Based on these esults, dose levels of 0, 100, 300 and 1000 mg/kg/day were selected for the current study.

Positive control:

No positive control was used in this study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, all animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day (except on days of receipt and necropsy where frequency was at least once daily).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed once daily, beginning during the first administration of the test material up to the day prior to necropsy. During the Dosing Period, observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals. Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.


Arena Observations:: All animals were observed once before the first administration of the test material and weekly thereafter during the Treatment Period.


BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Day 1 of treatment (prior to dosing) and weekly thereafter. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 Post Coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes (quantitatively measured per cage); Weekly except for males and females which were housed together for mating and for females without evidence of mating.

Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 Post Coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Regular basis throughout the study; Monitored by visual inspection of the water bottles. No quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane); Sampled from the retro-orbital sinus under anesthesia using isoflurane between 07.00 and 10.30 a.m
- Animals fasted: Males fasted overnight (maximum of 24 hours), females: No
- How many animals: 5/sex/group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Animals fasted: Males fasted overnight (maximum of 24 hours females non-fasted
- How many animals: 5/sex/group
- Parameters checked in table [No.3] were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes (Thyroid hormones)
- Time of blood sample collection: On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: Selected F0 animals (5/sex/dose) and Non-selected F0 animals (≤ 5/sex/group)

Blood samples (1.0 mL) were processed for serum, and serum was analyzed for Triiodothyronine (T3); Thyroxine (T4); and Thyroid stimulating hormone (TSH). After clotting and centrifugation, the serum was used for T3, T4 and TSH analysis. The serum derived from the F0-animals was divided in two aliquots. One aliquot was used for measurement of TSH using the IMMULITE® 1000 analyser and the other aliquot was used for measurement of T3 and T4 using LC-MS.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: F0-males: during Week 4 of treatment; F0-females: during the last week of lactation (i.e., PND 8).
- Dose groups that were examined: selected 5 males per group and selected 5 females per group for all dose groups
- Battery of functions tested: Hearing ability; Pupillary reflex; Static righting reflex; Fore- and hind-limb grip strength; and Locomotor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Tables 5 and 6)

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females.

Unscheduled Deaths/Euthanasia:

Female No. 51 (100 mg/kg/day) was found dead. A necropsy was conducted and specified tissues were saved.

Scheduled Euthanasia:

Animals surviving until scheduled euthanasia were weighed, and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full postmortem examination. Scheduled necropsies were conducted for males (which sired or failed to sire) following completion of the mating period (a minimum of 28 days of administration); on PND 14-16 for females which delivered; and post-coitum Day 25 for females (with evidence of mating) which failed to deliver (Nos. 49, 59, and 78).

All males were fasted overnight with a maximum of 24 hours before necropsy (water was available). F0 females were not fasted.

ORGAN WEIGHTS:
Organs identified in Tables 5 and 6 were weighed at necropsy for all scheduled euthanasia animals and females with total litter loss. Paired organs were weighed together and organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes (see Tables 7 and 8)

Representative samples of the tissues identified in Tables 7 and 8 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin as follows:

- Selected animals and unscheduled deaths (found dead): Tissues identified in Text Table 7 (except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
- Males that failed to sire (except for males which were selected) and females that failed to deliver pups: Cervix, epididymis. coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus, and vagina.
- Remaining animals: Gross lesions/masses.

Alpha-2u-globulin staining:
Two slides per animal (i.e., one selected control male, one selected control female and five selected Group 4 males) were analyzed for HRP-polymer based chromogenic immunostaining of alpha-2u-globulin.

Microscopic Evaluation:
For the testes of all selected males of Groups 1 and 4, and males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. All tissues defined under Histology were examined.

Optional endpoint(s):

Optional endpoints: Not specified

Statistics:

Please see 'Any other information on materials and methods incl. tables' for information on statistics.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed during the daily detailed clinical observations or during the weekly arena observations.

Salivation was observed post-dosing from the second week of the premating period and onwards in all male and female rats treated at 300 and 1000 mg/kg/day. Taking into account the nature and minor severity of the effect and its time of occurrence (i.e., after dosing), this sign was considered to
be a physiological response rather than a sign of systemic toxicity.

Any other clinical signs observed during the dosing period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related mortality was observed through the study period.

One female rat (No. 51) in the100 mg/kg/day dose group was found dead on Day 7 post-coitum (Day 26 of treatment). No clinical signs were observed prior to death, however, a slightly lower food consumption was noted over Days 4-7 post-coitum. Pathologic findings of note in this animal consisted of watery-clear content with accumulation of gray-white material in the thoracic cavity and microscopic acute bronchioalveolar inflammation with alveolar necrosis and foreign material in the lungs, which was related to the macroscopic finding of not collapsed lungs at necropsy. Moderate extramedullary hematopoiesis was seen in the spleen but considered secondary to the inflammation in the lung. The findings were considered to be gavage procedure-related and not treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gain of male and female rats in the 100 and 300 mg/kg/day dose groups were comparable to concurrent controls.

In male rats treated at 1000 mg/kg/day, body weight gain was slightly lower when compared to controls throughout the dosing period (occasionally statistically significant), resulting in a 4% lower final mean body weight at the end of the dosing period (not statistically significant).

In female rats treated at 1000 mg/kg/day, body weight gain was slightly lower at the end of the post-coitum period only, with a 4% lower mean body weight at the end of the post-coitum and lactation periods (all not statistically significant).

Given the small magnitude and transience, and in the absence of corroborative changes in food consumption during those periods, these effects on body weight (gain) were considered to be non-adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was unaffected by treatment in male rats at all dose levels and in female rats at doses up to 300 mg/kg/day.

In female rats treated at 1000 mg/kg/day, food consumption (before and after correction for body weight) was lower in the first week of the dosing period and in the last week of the lactation period when compared to control animals. Given the small magnitude and transience, the effects on food consumption were considered to be non-adverse.

In the absence of a dose-response trend, any other changes in food consumption (after correction for body weight) were considered to be unrelated to treatment, but rather a result of slightly lower body weights.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Monitored by visual inspection of the water bottles. No quantitative investigation was introduced as no effect was suspected.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological parameters in female rats were unaffected by treatment at all dose levels tested and up to 100 mg/kg/day in male rats.

In male rats, higher total white blood cell, neutrophil, and lymphocyte counts were observed at 1000 mg/kg/day (1.32, 1.28, and 1.33x of control, respectively, not statistically significant for neutrophil and lymphocyte counts). Higher red blood cell distribution width at 300 and 1000 mg/kg/day (1.06 and 1.10x of control, respectively), accompanied by higher reticulocyte count were also observed in male rats at 1000 mg/kg/day (1.17x of control, not statistically significant).

Considering the small magnitude of changes and in the absence of corroborative histopathology findings, the above changes were determined to be non-adverse.

Coagulation parameters in male and female rats remained unaffected by treatment through the study period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry parameters in male and fame rats were unaffected by treatment at 100 mg/kg/day.

Higher potassium levels were observed in male rats from 300 mg/kg/day onwards (1.11 and 1.16x of control at 300 and 1000 mg/kg/day, respectively) while higher creatinine levels were observed in male rats at 1000 mg/kg/day (1.35x of control).

Higher total bilirubin levels were observed in female rats at 1000 mg/kg/day (1.58x of control).

Considering the small magnitude of changes and in the absence of corroborative histopathology findings, the above changes were determined to be non-adverse.

Any other changes observed in clinical chemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Serum levels of T3 and TSH were considered unaffected by treatment at 100 mg/kg/day in male rats and at all dose levels in female rats. Serum levels of T4 were considered unaffected by treatment with the test material in either sex at all dose levels tested.

T3 levels were observed to be lower than the control mean in male rats at 300 and 1000 mg/kg/day (0.73 and 0.67x, respectively) with an associated increase in serum levels of TSH. Mean TSH values were 2.14 and 2.31x of control at 300 and 1000 mg/kg/day, respectively. The mean TSH values fell above the historical control range, while mean T3 values remained within the historical control range. In the absence of a relevant microscopic correlate in the thyroid gland, these findings were considered to be non-adverse.

The lower mean T3 level observed in female rats at 300 mg/kg/day (0.73x of control), was considered unrelated to treatment due to a general overlap in individual values.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex, and grip strength were observed to be normal in all examined animals.

Motor activity was unaffected by treatment up to 300 mg/kg/day. At 1000 mg/kg/day, the number of total movements was lower than the concurrent control mean in both male and female rats (0.81 and 0.76x of control, respectively; not statistically significant), without notable effect on ambulation’s. These findings were not considered to be an adverse effect on neurobehavior since these results were not supported by clinical observations or other functional observation tests (including ambulations), were slight in nature, and had no supportive morphological correlates in examined neuronal tissues.

All groups exhibited a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related higher liver weights (males and females), thyroid gland weight (males) and kidney weight (males) were observed.

Dose-related higher liver weights (absolute and/or relative to body weights) were observed in in male and female rats starting at 300 mg/kg/day. Based on the low magnitude of the weight difference and in the absence of any other additional microscopic degenerative findings or related enzyme changes, the liver findings were considered to be non-adverse.

Higher thyroid gland weights (relative to body weights) were observed in male rats at 1000 mg/kg/day. Based on nature and severity, the thyroid gland findings were considered to be non-adverse.

Higher kidney weights (absolute and relative to body weights) were observed in male rats at 1000 mg/kg/day. Higher kidney weights in male rats at 1000 mg/kg/day correlated to microscopic hyaline droplet nephropathy, which although treatment-related and adverse, was considered to be male rat-specific and not relevant to humans.

Any other differences, including those that reached statistical significance (higher absolute and relative prostate gland weight at 100 mg/kg/day and higher absolute and relative testes weight at 100 and 1000 mg/kg/day) were not considered to be treatment-related due to the direction of the change and lack of dose-related pattern.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross necropsy revealed enlarged thyroid glands (3/10 males), black-brown discoloration of the liver (3/10 males), enlarged kidneys (1/10 males) and enlarged liver (1/10 females) at 1000 mg/kg/day. These effects were considered to be treatment-related but based on the low magnitude of the weight difference, the low severity of the hepatocellular hypertrophy and the absence of any other additional microscopic degenerative findings or related enzyme changes, the liver and thyroid findings were considered to be non-adverse.

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic findings were observed in the liver, spleen, and thyroid gland of both sexes and in the kidney of male rats.

Liver: A dose-related increased incidence and severity in centrilobular hepatocellular hypertrophy (up to a slight degree) was observed in male rats starting at 100 mg/kg/day and in female rats (at minimal degree) starting at 300 mg/kg/day. Based on the low severity of the hepatocellular hypertrophy and in the absence of any other additional microscopic degenerative findings or related enzyme changes, the liver findings were considered to be non-adverse.

Spleen: An increased incidence and severity in extramedullary hematopoiesis was observed in the spleen of male (up to moderate degree) and female (up to slight degree) rats at 1000 mg/kg/day. This is a common finding in the rat and given the low magnitude and in the absence of changes in hematology, was considered to be non-adverse.

Throid: An increased incidence and/or severity of follicular cell hypertrophy (up to a slight degree) was observed in male and female rats starting at 300 mg/kg/day. Based on the nature and severity, the thyroid gland findings were considered to be non-adverse.

A dose-dependent increase in hyaline droplet accumulation was observed in male rats at 100 and 300 mg/kg/day progressing to hyaline droplet nephropathy (an increase in hyaline droplet accumulation, increased tubular basophilia and the presence of granular casts) up to a moderate degree in male rats at 1000 mg/kg/day. After immunohistochemistry using alpha 2u-globulin antibodies, the hyaline droplets stained positive for alpha 2u-globulin. The control male showed small alpha 2u-globulin dots in the proximal tubules and the control female was alpha 2u-globulin negative. Based on the degenerative nature of some of the kidney findings (granular casts), the hyaline droplet nephropathy observed at 1000 mg/kg/day was considered adverse. However, this effect is male rat-specific and not relevant to humans.

Any other observed microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no treatment-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Systemic Toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Formulation analysis:

 

Concentration analysis:

 

The concentrations analyzed in the formulations of Groups 2, 3, and 4 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90%-110% of target concentration).

 

No test material was detected in the Group 1 (control) formulation.

 

Homogeneity analysis:

 

The formulations of Groups 2 and 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

 

Table 9. Results of Formulation Analysis
Date of analysis	Concentration (mg/mL)			Recovery (%)
	Target	Nominal	Analyzed	Individual	Mean
2022-FEB-16	25	27.2	25.0	92	92
		25.8	24.9	97
		25.2	22.3	88
	250	249	264	106	108
		250	283	113
		253	263	104

 

Table 10. Body Weights (grams) Result Summary
		Group 1 (Control – 0 mg/kg/day)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
Males
Pre-mating
Day 1 Week 1	Mean	319	317	328	321
	SD	8.7	14.3	8.9	10.6
	N	10	10	10	10
Day 8 Week 2	Mean	348	344	353	341
	SD	10.6	13.7	10.0	11.0
	N	10	10	10	10
Mating Period
Day 1 Week 1	Mean	367	364	373	359
	SD	12.0	17.4	15.5	12.8
	N	10	10	10	10
Day 8 Week 2	Mean	378	375	384	367
	SD	14.8	20.1	16.5	14.2
	N	10	10	10	10
Day 15 Week 3	Mean	395	391	398	381
	SD	15.1	20.6	18.2	16.2
	N	10	10	10	10
Females
Pre-mating
Day 1 Week 1	Mean	236	226	223	232
	SD	15.0	13.8	7.3	8.5
	N	10	10	10	10
Day 8 Week 2	Mean	234	229	225	235
	SD	15.4	16.0	6.4	8.1
	N	10	10	10	10
Mating Period
Day 1 Week 1	Mean	236	228	231	238
	SD	15.9	12.6	7.8	6.7
	N	10	10	10	10
Day 8 Week 2	Mean	251	---
	SD	---	---
	N	1	0 x
Day 15 Week 3	Mean		---
	SD		---
	N		0 x
Day 22 Week 4	Mean		---
	SD		---
	N		0 x
Post Coitum
Day 0	Mean	239	235	233	239
	SD	12.6	14.3	6.0	11.7
	N	9	8	10	9
Day 4	Mean	252	250	245	251
	SD	13.2	17.2	7.0	12.1
	N	9	8	10	9
Day 7	Mean	259	255	253	259
	SD	12.9	17.8	9.1	13.0
	N	9	7	10	9
Day 11	Mean	275	269	269	273
	SD	13.6	17.7	10.6	14.9
	N	9	7	10	9
Day 14	Mean	286	281	278	282
	SD	13.9	19.1	11.5	15.0
	N	9	7	10	9
Day 17	Mean	310	305	303	302
	SD	14.5	19.2	15.4	19.7
	N	9	7	10	9
Day 20	Mean	349	345	341	336
	SD	19.6	24.7	22.9	28.9
	N	9	7	10	9
Lactation
Day 1	Mean	275	265	266	267
	SD	17.8	18.1	10.8	14.0
	N	9	8	10	9
Day 4	Mean	280	276	277	275
	SD	14.0	15.3	12.4	14.2
	N	9	8	10	9
Day 7	Mean	288	283	284	281
	SD	12.4	17.7	10.4	14.3
	N	9	8	10	9
Day 13	Mean	303	294	296	291
	SD	16.1	18.1	12.9	16.3
	N	9	8	10	9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanations for excluded data are listed in the tables of the individual values presented in the final study report available with the registrant/CRO

 

Table 11. Body Weight Gain (%) Result Summary
		Group 1 (Control – 0 mg/kg/day)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
Males
Pre-mating
Day 1 Week 1	Mean	0	0	0	0
	SD	0.0	0.0	0.0	0.0
	N	10	10	10	10
Day 8 Week 2	Mean	9	9	8	6*
	SD	1.9	2.7	2.1	1.7
	N	10	10	10	10
Mating Period
Day 1 Week 1	Mean	15	15	14	12
	SD	2.7	3.2	4.2	2.2
	N	10	10	10	10
Day 8 Week 2	Mean	19	18	17	15
	SD	3.8	4.1	4.4	2.8
	N	10	10	10	10
Day 15 Week 3	Mean	24	23	22	19*
	SD	3.6	4.1	4.7	3.5
	N	10	10	10	10
Females
Pre-mating
Day 1 Week 1	Mean	0	0	0	0
	SD	0.0	0.0	0.0	0.0
	N	10	10	10	10
Day 8 Week 2	Mean	-1	1	1	1
	SD	3.0	1.8	2.4	2.6
	N	10	10	10	10
Mating Period
Day 1 Week 1	Mean	0	1	3	2
	SD	3.5	3.0	2.7	2.8
	N	10	10	10	10
Day 8 Week 2	Mean	14
	SD	---	---
	N	1 x	0 x
Day 15 Week 3	Mean		---
	SD		---
	N		0 x
Day 22 Week 4	Mean		---
	SD		---
	N		0 x
Post Coitum
Day 0	Mean	0	0	0	0
	SD	0.0	0.0	0.0	0.0
	N	9	8	10	9
Day 4	Mean	5	6	5	5
	SD	1.3	1.9	1.5	2.5
	N	9	8	10	9
Day 7	Mean	8	10	9	9
	SD	1.8	2.3	2.5	2.8
	N	9	7	10	9
Day 11	Mean	15	16	15	14
	SD	1.2	2.6	3.3	3.7
	N	9	7	10	9
Day 14	Mean	20	21	19	18
	SD	2.0	3.2	3.7	3.1
	N	9	7	10	9
Day 17	Mean	30	31	30	26
	SD	1.8	3.5	5.5	4.4
	N	9	7	10	9
Day 20	Mean	46	48	46	41
	SD	3.9	4.8	8.8	7.1
	N	9	7	10	9
Lactation
Day 1	Mean	0	0	0	0
	SD	0.0	0.0	0.0	0.0
	N	9	8	10	9
Day 4	Mean	2	4	4	3
	SD	4.2	3.2	2.7	2.9
	N	9	8	10	9
Day 7	Mean	5	7	7	5
	SD	4.3	3.1	2.5	3.4
	N	9	8	10	9
Day 13	Mean	10	11	11	9
	SD	5.9	3.2	3.3	3.9
	N	9	8	10	9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanations for excluded data are listed in the tables of the individual values presented in the final report available with the registrant/CRO

 

Table 12. Food Consumption (g/animal/day) Result Summary
		Group 1 (Control – 0 mg/kg/day)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
Males
Pre-mating
Days 1-8 Weeks 1-2	Mean	24	23	24	22
	SD	0.7	0.7	0.2	1.0
	N (Cage)	2	2	2	2
Days 8-15 Weeks 2-3	Mean	23	22	24	23
	SD	0.5	0.3	0.3	0.2
	N (Cage)	2	2	2	2
Mean of means over Pre-mating	Mean	23	23	24	22
Mating Period
Days 1-8 Weeks 1-2	Mean	22	23	23	24
	SD	0.8	1.2	0.2	1.0
	N (Cage)	2	2	2	2
Days 8-15 Weeks 2-3	Mean	21	22	22	23
	SD	0.3	0.4	0.5	0.7
	N (Cage)	2	2	2	2
Mean of means over Mating Period	Mean	22	22	22	23
Females
Pre-mating
Days 1-8 Weeks 1-2	Mean	15	16	15	14*
	SD	0.1	0.2	0.3	0.3
	N (Cage)	2	2	2	2
Days 8-15 Weeks 2-3	Mean	15	15	15	16
	SD	0.7	0.6	0.4	0.2
	N (Cage)	2	2	2	2
Mean of means over Pre-mating	Mean	15	15	15	15
Mating Period
Days 1-8 Weeks 1-2	Mean		---
	SD		---
	N (Cage)		0
Post-Coitum
Days 0-4	Mean	20	20	20	21
	SD	1.7	1.9	1.9	1.6
	N	9	8	10	9
Days 4-7	Mean	18	17	18	19
	SD	1.3	2.9	1.8	1.2
	N	9	8	10	9
Days 7-11	Mean	18	18	18	19
	SD	1.4	1.8	1.9	1.1
	N	9	7	10	9
Days 11-14	Mean	19	20	19	21
	SD	1.6	2.8	2.1	2.0
	N	9	7	10	9
Days 14-17	Mean	20	21	21	22
	SD	1.3	2.4	2.0	2.2
	N	9	7	10	9
Days 17-20	Mean	22	23	23	24
	SD	1.9	2.5	3.1	2.4
	N	9	7	10	9
Mean of means		19	20	20	21
Lactation
Days 1-4	Mean	26	28	29	28
	SD	2.3	4.7	5.0	4.8
	N	9	8	10	9
Days 4-7	Mean	38	39	38	35
	SD	1.9	4.7	3.5	6.1
	N	9	8	10	9
Days 7-13	Mean	52	51	48	43*
	SD	2.9	5.9	5.2	9.2
	N	9	8	10	9
Mean of means		39	39	38	35

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanations for excluded data are listed in the tables of the individual values presented in the final report available with the registrant/CRO.

 

Table 13. Relative Food Consumption (g/Kg body weight/day) Result Summary
		Group 1 (Control – 0 mg/kg/day)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
Males
Pre-mating
Days 1-8 Weeks 1-2	Mean	68	68	68	64
	SD	0.8	2.6	0.2	2.2
	N (Cage)	2	2	2	2
Days 8-15 Weeks 2-3	Mean	65	65	67	67
	SD	0.3	1.5	1.3	1.4
	N (Cage)	2	2	2	2
Mean of means over Pre-mating	Mean	67	66	67	65
Mating Period
Days 1-8 Weeks 1-2	Mean	58	61	59	64
	SD	2.9	1.9	0.8	3.3
	N (Cage)	2	2	2	2
Days 8-15 Weeks 2-3	Mean	54	56	54	60*
	SD	1.3	0.0	1.4	2.3
	N (Cage)	2	2	2	2
Mean of means over Mating Period	Mean	56	59	57	62
Females
Pre-mating
Days 1-8 Weeks 1-2	Mean	64	68**	67*	59**
	SD	0.9	0.2	0.9	0.3
	N (Cage)	2	2	2	2
Days 8-15 Weeks 2-3	Mean	63	65	66	70*
	SD	2.0	1.7	1.1	1.1
	N (Cage)	2	2	2	2
Mean of means over Pre-mating	Mean	63	67	67	64
Mating Period
Days 1-8 Weeks 1-2	Mean		---
	SD		---
	N (Cage)		0 x
Post-Coitum
Days 0-4	Mean	78	80	83	85
	SD	7.7	8.3	6.4	5.7
	N	9	8	10	9
Days 4-7	Mean	70	70	73	75
	SD	6.6	7.8	5.4	4.2
	N	9	7	10	9
Days 7-11	Mean	64	68	65	68
	SD	4.2	6.7	5.0	1.2
	N	9	7	10	9
Days 11-14	Mean	67	73	69	75*
	SD	4.8	7.9	5.9	6.7
	N	9	7	10	9
Days 14-17	Mean	64	69	68	72**
	SD	3.7	6.9	3.6	4.6
	N	9	7	10	9
Days 17-20	Mean	62	67	67	71**
	SD	5.4	6.6	5.0	3.5
	N	9	7	10	9
Mean of means		68	71	71	74
Lactation
Days 1-4	Mean	93	102	106	101
	SD	8.4	15.2	14.6	14.8
	N	9	8	10	9
Days 4-7	Mean	133	136	132	124
	SD	5.5	15.2	8.4	18.9
	N	9	8	10	9
Days 7-13	Mean	171	173	162	148*
	SD	6.1	16.3	13.6	27.2
	N	9	8	10	9
Mean of means		132	137	133	125

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanations for excluded data are listed in the tables of the individual values presented in the final report available with the registrant/CRO.

 

Table 14. Functional Observations Summary
		Group 1 (Control – 0 mg/kg/day)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
Males
End of Treatment
Hearing Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Pupil L Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Pupil R Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Static R Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Grip Fore gram	Mean	1574	1485	1551	1474
	SD	287	293	197	197
	N	5	5	5	5
Grip Hind gram	Mean	847	672	743	710
	SD	153	167	146	76
	N	5	5	5	5
Females
Hearing Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Pupil L Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Pupil R Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Static R Score 0/1	Median	0	0	0	0
	N	5	5	5	5
Grip Fore gram	Mean	1191	979	1200	1086
	SD	147	176	128	208
	N	5	5	5	5
Grip Hind gram	Mean	496	431	471	438
	SD	60	86	58	82
	N	5	5	5	5

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 15. Summary of Select Hemaology Values: F0 Generation (Males)
Day: 30 Relative to Start Date (Males)		Reporting Hematology
		WBC (10^9/L)	MONO (10^9/L)	RBC (10^12/L)	RDWG (%)
Statistical Test		[G]	[G]	[G]	[G]
Group 1 (Control – 0 mg/kg/day)	Mean	7.992	0.116	8.880	12.24
	SD	1.173	0.021	0.331	0.27
	N	5	5	5	5
Group 2 (100 mg/kg/day)	Mean	7.584	0.110	8.242*	12.12
	SD	1.693	0.035	0.487	0.33
	N	5	5	5	5
	tCtrl	0.95	0.95	0.93	0.99
Group 3 (300 mg/kg/day)	Mean	9.044	0.182*	8.848	12.94*
	SD	1.276	0.058	0.208	0.30
	N	5	5	5	5
	tCtrl	1.13	1.57	1.00	1.06
Group 4 (1000 mg/kg/day)	Mean	10.538*	0.130	8.566	13.42**
	SD	1.716	0.037	0.354	0.48
	N	5	5	5	5
	tCtrl	1.32	1.12	0.96	1.10

[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

WBC: White blood cell count

MONO: Monocyte count

RBC: Red blood cell count

RDWG: Red blood cell distribution width

 

Table 16. Summary of Select Biochemistry Values: F0 Generation (Males)
Day: 30 Relative to Start Date (Males)		Reporting Biochemistry
		CREAT (umol/L)	K (mmol/L)	T3 (ng/mL)	T4 (ng/mL)	TSH (mU/L)
Statistical Test		[G1]	[G]	[G1]	[G1]	[G]
Group 1 (Control – 0 mg/kg/day)	Mean	27.4	4.02	0.447	48.55	0.2622
	SD	1.5	0.15	0.102	6.66	0.1379
	N	5	5	10	10	10
Group 2 (100 mg/kg/day)	Mean	31.2	4.20	0.398	55.40	0.3797
	SD	1.3	0.21	0.093	8.85	0.3177
	N	5	5	10	10	10
	tCtrl	1.14	1.05	0.89	1.14	1.45
Group 3 (300 mg/kg/day)	Mean	30.8	4.45*	0.328**	53.56	0.5601*
	SD	2.6	0.21	0.063	8.91	0.2386
	N	5	5	10	10	10
	tCtrl	1.13	1.11	0.73	1.10	2.14
Group 4 (1000 mg/kg/day)	Mean	36.9**	4.67*	0.300**	45.40	0.6059
	SD	5.0	0.61	0.071	6.86	0.4265
	N	5	5	10	10	10
	tCtrl	1.35	1.16	0.67	0.94	2.31

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Kruskal-Wallis & Dunn: ** = p ≤ 0.01

CREAT: Creatinine

K: Potassium

T3: Triiodothyronine

T4: Thyroxine

TSH: Thyroid stimulating hormone

 

Table 17. Summary of Select Biochemistry Values: F0 Generation (Females)
Day: 14 Relative to Start Date (Females)		Reporting Biochemistry
		TBIL (umol/L)	K (mmol/L)	CL (mmol/L)	T3 (ng/mL)
Statistical Test		[G]	[G]	[G]	[G]
Group 1 (Control – 0 mg/kg/day)	Mean	1.68	5.47	107.6	0.277
	SD	0.66	0.44	1.7	0.044
	N	5	5	5	10
Group 2 (100 mg/kg/day)	Mean	1.90	5.07	104.2*	0.231
	SD	0.42	0.34	2.6	0.038
	N	5	5	5	8
	tCtrl	1.13	0.93	0.97	0.84
Group 3 (300 mg/kg/day)	Mean	1.94	4.77*	108.0	0.203**
	SD	0.38	0.33	2.3	0.043
	N	5	5	5	10
	tCtrl	1.15	0.87	1.00	0.73
Group 4 (1000 mg/kg/day)	Mean	2.66*	4.89*	105.0	0.226
	SD	0.34	0.29	1.4	0.060
	N	5	5	5	9
	tCtrl	1.58	0.89	0.98	0.82

[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

TBIL: Total bilirubin

K: Potassium

CL: Chloride

T3: Triiodothyronine

 

Table 18. Summary of Macroscopic Findings (F0 Generation)
	Group 1 (Control – 0 mg/kg/day)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
Males
End of Treatment
Animals examined	10	10	10	10
Animals without findings	9	9	9	4
Animals affected	1	1	1	6
Liver
Discolouration	0	0	0	3
Kidneys
Pelvic dilation	0	0	0	1
Enlarged	0	0	0	1
Epididymides
Nodule(s)	0	1	0	0
Coagulating Glands
Enlarged	0	0	1	0
Seminal Vesicles
Enlarged	0	0	1	0
Thyroid Gland
Enlarged	0	0	0	3
Reduced in size	1	0	0	0
Females
Intercurrent Death
Animals examined		1
Animals affected		1
General observations
Beginning autolysis		1
Lungs
Not collapsed		1
Body Cavities
Thoracic cavity material accumulation many,		1
Contents:		1
End of Treatment
Animals examined	9	8	10	9
Animals without findings	7	7	10	8
Animals affected	2	1	0	1
Liver
Enlarged	0	0	0	1
Thymus
Focus/foci	1	1	0	0
Mesenteric lymph node
Focus/foci	1	0	0	0
Non Pregnant
Animals examined	1	1	-	1
Animals without findings	1	1	-	1

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

Table 19. Select Organ Weights (grams) Summary
End of Treatment		Group 1 (Control – 0 mg/kg/day)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
Males
Liver (Gram)	Mean	9.36	9.46	10.70*	11.69**
	SD	0.50	0.63	0.66	1.23
	N	5	5	5	5
Kidneys (Gram)	Mean	2.38	2.53	2.63	2.79**
	SD	0.15	0.13	0.17	0.24
	N	5	5	5	5
Testes (Gram)	Mean	3.36	3.69*	3.65	3.82**
	SD	0.21	0.36	0.34	0.26
	N	10	10	10	10
Prostate Gland (Gram)	Mean	0.830	0.944*	0.917	0.834
	SD	0.052	0.117	0.098	0.115
	N	10	10	10	10
Females
Liver (Gram)	Mean	12.09	12.69	13.45	14.48*
	SD	1.31	1.17	0.82	1.97
	N	5	5	5	5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 20. Select Organ/Body Weight Ratios (%) Summary
End of Treatment		Group 1 (Control – 0 mg/kg/day)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
Males
Liver (%)	Mean	2.52	2.55	2.88**	3.33**
	SD	0.03	0.18	0.12	0.21
	N	5	5	5	5
Thyroids (%)	Mean	0.0042	0.0041	0.0046	0.0056*
	SD	0.0010	0.0006	0.0010	0.0017
	N	10	9	9	10
Kidneys (%)	Mean	0.64	0.68	0.71	0.80**
	SD	0.04	0.04	0.06	0.05
	N	5	5	5	5
Testes (%)	Mean	0.91	1.01*	0.99	1.09**
	SD	0.04	0.12	0.09	0.08
	N	10	10	10	10
Prostate Gland (%)	Mean	0.225	0.258*	0.248	0.237
	SD	0.019	0.031	0.026	0.032
	N	10	10	10	10
Females
Liver (%)	Mean	4.11	4.32	4.67*	5.02**
	SD	0.25	0.19	0.19	0.49
	N	5	5	5	5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Applicant's summary and conclusion

Conclusions:

Based on the alpha 2µ-globulin nephropathy observed in the kidneys of male rats at 1000 mg/kg/day, the systemic toxicity No Observed Adverse Effect Level (NOAEL) for Petroleum diesel/gas oil fraction, co-processed with renewable hydrocarbons derived from thermally cracked plastics was determined to be 300 mg/kg/day in male rats. However, this effect is male rat specific and of no relevance to humans. Therefore, not considering the alpha 2µ-globulin nephropathy, in the absence of any other treatment-related systemic toxicity effects observed through the study period, the systemic toxicity NOAEL was determined to be ≥1000 mg/kg/day in male and female rats.

Executive summary:

A key OECD Guideline 422 combined 28-Day repeated dose toxicity study with the reproduction / developmental toxicity screening test was conducted to determine the potential toxic effects of the test material (Petroleum diesel/gas oil fraction, co-processed with renewable hydrocarbons derived from thermally cracked plastics (EC 941-803-4)) when administered orally to Wistar Han rats for a minimum of 28 days.

 

The test material in a corn oil vehicle was administered to Wistar Han rats (10/sex/dose) at doses of 0, 100, 300, or 1000 mg/kg/day. Male rats were exposed 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy. Female rats were exposed 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, measurement of thyroid hormones T3, T4 and TSH (F0 males and females), gross necropsy findings, organ weights and histopathologic examinations were parameters evaluated in the study.

 

No treatment-related mortality or clinical signs of toxicity were observed during the daily detailed clinical observations or during the weekly arena observations. Salivation was observed post-dosing from the second week of the premating period and onwards in all male and female rats treated at 300 and 1000 mg/kg/day. Taking into account the nature and minor severity of the effect and its time of occurrence (i.e., after dosing), this sign was considered to be a physiological response rather than a sign of systemic toxicity.

 

Body weights and body weight gain of male and female rats in the 100 and 300 mg/kg/day dose groups were comparable to concurrent controls. In male rats treated at 1000 mg/kg/day, body weight gain was slightly lower when compared to controls throughout the dosing period (occasionally statistically significant), resulting in a 4% lower final mean body weight at the end of the dosing period (not statistically significant). In female rats treated at 1000 mg/kg/day, body weight gain was slightly lower at the end of the post-coitum period only, with a 4% lower mean body weight at the end of the post-coitum and lactation periods (all not statistically significant). Given the small magnitude and transience, and in the absence of corroborative changes in food consumption during those periods, these effects on body weight (gain) were considered to be non-adverse.

 

Food consumption before or after correction for body weight was unaffected by treatment in male rats at all dose levels and in female rats at doses up to 300 mg/kg/day. In female rats treated at 1000 mg/kg/day, food consumption (before and after correction for body weight) was lower in the first week of the dosing period and in the last week of the lactation period when compared to control animals. Given the small magnitude and transience, the effects on food consumption were considered to be non-adverse. In the absence of a dose-response trend, any other changes in food consumption (after correction for body weight) were considered to be unrelated to treatment, but rather a result of slightly lower body weights.

 

Hematological parameters in female rats were unaffected by treatment at all dose levels tested and up to 100 mg/kg/day in male rats. In male rats, higher total white blood cell, neutrophil, and lymphocyte counts were observed at 1000 mg/kg/day (1.32, 1.28, and 1.33x of control, respectively, not statistically significant for neutrophil and lymphocyte counts). Higher red blood cell distribution width at 300 and 1000 mg/kg/day (1.06 and 1.10x of control, respectively), accompanied by higher reticulocyte count were also observed in male rats at 1000 mg/kg/day (1.17x of control, not statistically significant). Considering the small magnitude of changes and in the absence of corroborative histopathology findings, the above changes were determined to be non-adverse. Coagulation parameters in male and female rats remained unaffected by treatment through the study period.

 

Clinical chemistry parameters in male and fame rats were unaffected by treatment at 100 mg/kg/day. Higher potassium levels were observed in male rats from 300 mg/kg/day onwards (1.11 and 1.16x of control at 300 and 1000 mg/kg/day, respectively) while higher creatinine levels were observed in male rats at 1000 mg/kg/day (1.35x of control). Higher total bilirubin levels were observed in female rats at 1000 mg/kg/day (1.58x of control). Considering the small magnitude of changes and in the absence of corroborative histopathology findings, the above changes were determined to be non-adverse. Any other changes observed in clinical chemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

 

Serum levels of T3 and TSH were considered unaffected by treatment at 100 mg/kg/day in male rats and at all dose levels in female rats. Serum levels of T4 were considered unaffected by treatment with the test material in either sex at all dose levels tested. T3 levels were observed to be lower than the control mean in male rats at 300 and 1000 mg/kg/day (0.73 and 0.67x, respectively) with an associated increase in serum levels of TSH. Mean TSH values were 2.14 and 2.31x of control at 300 and 1000 mg/kg/day, respectively. The mean TSH values fell above the historical control range, while mean T3 values remained within the historical control range. In the absence of a relevant microscopic correlate in the thyroid gland, these findings were considered to be non-adverse. The lower mean T3 level observed in female rats at 300 mg/kg/day (0.73x of control), was considered unrelated to treatment due to a general overlap in individual values.

 

Hearing ability, pupillary reflex, static righting reflex, and grip strength were observed to be normal in all examined animals. Motor activity was unaffected by treatment up to 300 mg/kg/day. At 1000 mg/kg/day, the number of total movements was lower than the concurrent control mean in both male and female rats (0.81 and 0.76x of control, respectively; not statistically significant), without notable effect on ambulation’s. These findings were not considered to be an adverse effect on neurobehavior since these results were not supported by clinical observations or other functional observation tests (including ambulations), were slight in nature, and had no supportive morphological correlates in examined neuronal tissues. All groups exhibited a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

 

Gross necropsy revealed enlarged thyroid glands (3/10 males), black-brown discoloration of the liver (3/10 males), enlarged kidneys (1/10 males) and enlarged liver (1/10 females) at 1000 mg/kg/day. These effects were considered to be treatment-related but based on the low magnitude of the weight difference, the low severity of the hepatocellular hypertrophy and the absence of any other additional microscopic degenerative findings or related enzyme changes, the liver and thyroid findings were considered to be non-adverse. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

 

Treatment-related higher liver weights (males and females), thyroid gland weight (males) and kidney weight (males) were observed. Dose-related higher liver weights (absolute and/or relative to body weights) were observed in in male and female rats starting at 300 mg/kg/day. Based on the low magnitude of the weight difference and in the absence of any other additional microscopic degenerative findings or related enzyme changes, the liver findings were considered to be non-adverse. Higher thyroid gland weights (relative to body weights) were observed in male rats at 1000 mg/kg/day. Based on nature and severity, the thyroid gland findings were considered to be non-adverse. Higher kidney weights (absolute and relative to body weights) were observed in male rats at 1000 mg/kg/day. Higher kidney weights in male rats at 1000 mg/kg/day correlated to microscopic hyaline droplet nephropathy, which although treatment-related and adverse, was considered to be male rat-specific and not relevant to humans. Any other differences, including those that reached statistical significance (higher absolute and relative prostate gland weight at 100 mg/kg/day and higher absolute and relative testes weight at 100 and 1000 mg/kg/day) were not considered to be treatment-related due to the direction of the change and lack of dose-related pattern.

 

Treatment-related microscopic findings were observed in the liver, spleen, and thyroid gland of both sexes and in the kidney of male rats. A dose-related increased incidence and severity in centrilobular hepatocellular hypertrophy (up to a slight degree) was observed in male rats starting at 100 mg/kg/day and in female rats (at minimal degree) starting at 300 mg/kg/day. Based on the low severity of the hepatocellular hypertrophy and in the absence of any other additional microscopic degenerative findings or related enzyme changes, the liver findings were considered to be non-adverse. An increased incidence and severity in extramedullary hematopoiesis was observed in the spleen of male (up to moderate degree) and female (up to slight degree) rats at 1000 mg/kg/day. This is a common finding in the rat and given the low magnitude and in the absence of changes in hematology, was considered to be non-adverse. An increased incidence and/or severity of follicular cell hypertrophy (up to a slight degree) was observed in male and female rats starting at 300 mg/kg/day. Based on the nature and severity, the thyroid gland findings were considered to be non-adverse.

 

A dose-dependent increase in hyaline droplet accumulation was observed in male rats at 100 and 300 mg/kg/day progressing to hyaline droplet nephropathy (an increase in hyaline droplet accumulation, increased tubular basophilia and the presence of granular casts) up to a moderate degree in male rats at 1000 mg/kg/day. After immunohistochemistry using alpha 2u-globulin antibodies, the hyaline droplets stained positive for alpha 2u-globulin. The control male showed small alpha 2u-globulin dots in the proximal tubules and the control female was alpha 2u-globulin negative. Based on the degenerative nature of some of the kidney findings (granular casts), the hyaline droplet nephropathy observed at 1000 mg/kg/day was considered adverse. However, this effect is male rat-specific and not relevant to humans.

 

Based on the alpha 2µ-globulin nephropathy observed in the kidneys of male rats at 1000 ,g/kg/day, the systemic toxicity No Observed Adverse Effect Level (NOAEL) for Petroleum diesel/gas oil fraction, co-processed with renewable hydrocarbons derived from thermally cracked plastics was determined to be 300 mg/kg/day in male rats. However, this effect is male rat specific and of no relevance to humans. Therefore, not considering the alpha 2µ-globulin nephropathy, in the absence of any other treatment-related systemic toxicity effects observed through the study period, the systemic toxicity NOAEL was determined to be ≥1000 mg/kg/day in male and female rats.